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Amarantin is the predominant seed storage protein from amaranth. It shows a high content of essential amino acids, making this protein important from a nutritional viewpoint. The protein has two disulfide linked subunits: acidic and basic. Acidic subunit has the potential as a functional and nutraceutical protein, and it is structurally a good candidate for modification. In order to improve its functionality, the primary structure was modified in the third variable region of globulins 11S, by inserting four Val-Tyr antihypertensive peptides in tandem. The designed plasmid was expressed in Escherichia coli Origami (DE3) and then the expressed protein was purified. Mass spectrometry analysis was used to corroborate the identity of the protein by peptide mass fingerprinting; also, the modified peptide was fragmented and sequenced by mass spectrometry, corroborating thus the inserted residues. The hydrolyzed protein showed a high inhibitory activity of the angiotensin converting enzyme (IC(50) 0.064 mg ml(-1)); it was nearly eightfold more active than the nonmodified protein. In spite that the nonmodified subunit is less active, its activity is comparable with other hydrolyzed proteins reported as high active inhibitors. The expressed and purified subunit after its engineered modification, may be useful for preventing hypertension and for other medical purposes., (Copyright 2010 Elsevier B.V. All rights reserved.)

Some studies aimed at revealing the relationship between protein structure and their functional properties. However, the majority of these reports have been carried out using protein isolates. There are limited reports on the possible relationship between the functional properties and the structure of a purified protein. In this work the amaranth 11S globulin acidic subunit (AAC) and five mutations of the same protein that were modified in their variable regions with antihypertensive peptides (VYVYVYVY and RIPP), were analyzed at two ionic strength (2.9 and 17.6 g/L NaCl) and pH (3.0–7.0). Results revealed better solubility for the proteins mutated at the terminal ends (AACM.1 and AACM.4) and lower solubility for the protein inserted with RIPP peptide. Spectroscopy studies revealed an increase of β-sheet structure at high salt concentration for all proteins. It was also observed that salt concentration acted as a modulator, which allowed a better foam features for all modified proteins limiting movement of side chains and reducing red-shifted displacement of λmax. All proteins showed foam capacity ranging from 76 to 93% although foam stability was twofold better for modified proteins than for AAC at high salt concentration. This study allowed better understanding about the structural changes that influence the foaming properties of engineered proteins. [ABSTRACT FROM AUTHOR]

Featured Application: This work introduces a novel way of designing proteins for the treatment of diseases, especially metabolic syndrome. Metabolic syndrome comprises a cluster of diseases like hypertension, dyslipidemia, and insulin resistance, among others. Its treatment is based on lifestyle modification; however, this treatment often fails to improve metabolic syndrome indicators over the long term. In this work, sequences of some representative vegetable proteins were explored to find bioactive peptides with activity toward metabolic disorders of metabolic syndrome. Five proteins, i.e., legumin (chickpea), glutelin type A-2 (chickpea), glutelin type B-2 (rice), prolamin PPROL 17 (maize), and glutelin (rice) revealed a high potential to be effective against metabolic syndrome. We designed and evaluated in silico modifications to their amino acid sequence to release bioactive peptides after simulating gastrointestinal digestion (SGD). The approach presented here allows the design of proteins that could combat metabolic syndrome, for later production and study. In the future, these proteins can be used as functional foods. [ABSTRACT FROM AUTHOR]

Metabolic syndrome is a severe public health issue characterized by multiple metabolic disturbances. Current treatments prescribe a particular drug for each of them, producing multiple side effects. As a first step towards a more integral approach, we applied our recently described methodology to design single proteins, based in the Concanavalin B scaffold (1CNV), that contain several bioactive peptides (BPs), including antioxidant and lipid-lowering activities as well as inhibitors of dipeptidyl peptidase IV (DPPIV) and the angiotensin converting enzyme. Modified Concanavalin (CNV44), the designed protein that showed the best in silico properties, was expressed in high yields in E. coli and purified to homogeneity. After in vitro digestion with gastrointestinal enzymes, all the biological activities tested where higher in CNV44 when compared to the non-modified protein 1CNV, or to other previous reports. The results presented here represent the first in vitro evidence of a modified protein with the potential to treat metabolic syndrome and open the venue for the design of proteins to treat other non-communicable diseases. [ABSTRACT FROM AUTHOR]

Background: Proteins are often used in foods as ingredients to provide desirable appearance, texture, or stability. It is commonly used as gelling, emulsifiers, foaming, and thickeners. An awareness of its properties can be achieved by exploring the relationship between structural conformation adopted by a protein and its techno-functional properties. Here, we analyzed the techno-functional properties of the acidic subunit from amarantin AAC and modified variants AACM.1 and AACM.3 with bioactive peptides (VY 4x) insertion. The proteins were expressed at the fermenter level using Escherichia coli. The techno-functional properties were analyzed at different pH and compared with fresh white egg and sodium caseinate. Moreover, the structural characterization of proteins was done through infrared and Raman spectroscopies. Results: The techno-functional properties of all proteins studied were better at pH 7.5. Solubility and emulsifying activity of AAC and AACM.3 were similar to sodium caseinate, atpH5. AACM.3 showed similar foaming stability to the white egg, while at pH 7.5, both modified proteins showed higher foaming stability and were equivalent to the white egg. Spectroscopic techniques showed conformational differences between AAC and modified variants. AACM.3 showed higher unordered secondary content (59.6%) followed by AACM.1 (39.6%), suggesting AACM.3 adopted a structure as a molten globule by modifications done, which enhance its techno-functional properties. [ABSTRACT FROM AUTHOR]

Background: The 11S globulin from amaranth is the most abundant storage protein in mature seeds and is well recognized for its nutritional value. We used this globulin to engineer a new protein by adding a four valinetyrosine antihypertensive peptide at its C-terminal end to improve its functionality. The new protein was named AMR5 and expressed in the Escherichia coli BL21-CodonPlus(DE3)-RIL strain using a custom medium (F8PW) designed for this work. Results: The alternative medium allowed for the production of 652 mg/L expressed protein at the flask level, mostly in an insoluble form, and this protein was subjected to in vitro refolding. The spectrometric analysis suggests that the protein adopts a β/α structure with a small increment of α-helix conformation relative to the native amaranth 11S globulin. Thermal and urea denaturation experiments determined apparent Tm and C1/2 values of 50.4°C and 3.04 M, respectively, thus indicating that the antihypertensive peptide insertion destabilized the modified protein relative to the native one. AMR5 hydrolyzed by trypsin and chymotrypsin showed 14- and 1.3-fold stronger inhibitory activity against angiotensin I-converting enzyme (IC50 of 0.034 mg/mL) than the unmodified protein and the previously reported amaranth acidic subunit modified with antihypertensive peptides, respectively. Conclusion: The inserted peptide decreases the structural stability of amaranth 11S globulin and improves its antihypertensive activity. [ABSTRACT FROM AUTHOR]

Background: The acidic subunit of amarantin (AAC)--the predominant amaranth seed storage protein--has functional potential and its third variable region (VR) has been modified with antihypertensive peptides to improve this potential. Here, we modified the C-terminal in the fourth VR of AAC by inserting four VY antihypertensive peptides. This modified protein (AACM.4) was expressed in Escherichia coli. In addition, we also recombinantly expressed other derivatives of the amarantin protein. These include: unmodified amarantin acidic subunit (AAC); amarantin acidic subunit modified at the third VR with four VY peptides (AACM.3); and amarantin acidic subunit doubly modified, in the third VR with four VY peptides and in the fourth VR with the RIPP peptide (AACM.3.4). Results: E. coli BL21-CodonPlus (DE3)-RILwas the most favorable strain for the expression of proteins. After 6 h of induction, it showed the best recombinant protein titer. The AAC and AACM.4 were obtained at higher titers (0.56 g/L) while proteins modified in the third VR showed lower titers: 0.44 g/L and 0.33 g/L for AACM.3 and AACM.3.4, respectively. As these AAC variants were mostly expressed in an insoluble form, we applied a refolding protocol. This made it possible to obtain all proteins in soluble form. Modification of the VR 4 improves the thermal stability of amarantin acidic subunit; AAC manifested melting temperature (Tm) at 34°C and AACM.4 at 37.2°C. The AACM.3 and AACM.3.4 did not show transition curves. Conclusions: Modifications to the third VR affect the thermal stability of amarantin acidic subunit. [ABSTRACT FROM AUTHOR]